Use of GPRC5d single-domain antibody and humanized antibody thereof in construction of car-t cell

Abstract

Provided is the use of a GPRC5D single-domain antibody and a humanized antibody thereof in the construction of a CAR-T cell. The present invention specifically relates to a single-domain antibody targeting GPRC5D and a humanized antibody thereof, and tandem configurations of the antibodies. Further provided are a chimeric antigen receptor (CAR) molecule constructed by using the antibodies, a CAR-T cell and the use thereof, providing a new therapeutic option for patients with GPRC5D-positive multiple myeloma.

Description

Application of GPRC5D single-domain antibodies and their humanized antibodies in the construction of CAR-T cells Technical Field

[0001] This invention relates to the application of GPRC5D single-domain antibodies and their humanized antibodies in the construction of CAR-T cells, and relates to the fields of genetic engineering and antibody technology. Background Technology

[0002] Multiple myeloma (MM) is a malignant plasma cell disease. Its tumor cells originate from plasma cells in the bone marrow, which are the final functional stages of B lymphocyte development. Therefore, multiple myeloma can be classified as a type of B-cell lymphoma. The WHO classifies it as a type of B-cell lymphoma, called plasma cell myeloma / plasmacytoma. It is characterized by abnormal proliferation of plasma cells in the bone marrow accompanied by excessive production of monoclonal immunoglobulins or light chains (M protein). In rare cases, it can be a non-secreting type of MM that does not produce M protein. Multiple myeloma is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, and kidney damage. Due to the suppressed production of normal immunoglobulins, it is prone to various bacterial infections.

[0003] Currently, the main treatment for MM is BCMA (B-cell maturation antigen) targeting therapy. However, for MM patients who are BCMA negative or have low BCMA expression, relapse still occurs after receiving BCMA-targeted CAR-T cell therapy, indicating target escape. To alleviate BCMA escape-mediated relapse, finding better specific MM targets (such as GPRC5D) or simultaneously targeting other antigen targets (such as CD3, CD19, CD38, GPRC5D, etc.) is crucial. GPRC5D is likely to become a popular candidate target for the next treatment of MM.

[0004] GPRC5D (G protein-coupled receptor C5 family subtype D) is an atypical class C orphan G protein-coupled receptor, belonging to the 7-transmembrane protein family. In recent years, GPRC5D has become a highly attractive target for the treatment of multiple myeloma (MM). This is mainly because GPRC5D is highly expressed on the surface of multiple myeloma cells, while its expression in normal tissues is very limited. Therefore, this invention aims to provide antibody sequences targeting GPRC5D and their application in CAR-T and immune cell construction, thereby providing new treatment strategies for patients who have failed existing therapies or have relapsed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a class of single-domain antibodies targeting GPRC5D and humanized antibodies thereof, as well as the tandem configuration of the antibodies, and also to provide chimeric antigen receptor (CAR) molecules constructed using the antibodies, CAR-T cells, and their uses.

[0006] Specifically, the present invention includes the following:

[0007] The first aspect of the present invention provides an antibody targeting and binding to GPRC5D, said antibody having the amino acid sequences of the three CDR regions shown in SEQ ID NO:1, 2, 3; SEQ ID NO:5, 6, 7; SEQ ID NO:9, 10, 11; SEQ ID NO:13, 14, 15; SEQ ID NO:9, 20, 21; SEQ ID NO:23, 24, 25; SEQ ID NO:27, 28, 25; SEQ ID NO:30, 31, 32, or having amino acid sequences corresponding to those shown in SEQ ID NO:1, 2, 3; SEQ ID NO:5, 6, 7; SEQ ID NO:9, 10, 11; SEQ ID NO:13, 14, 15; SEQ ID NO:9, 20, 21; SEQ ID NO:23, 24, 25; SEQ ID NO:27, 28, 25; SEQ ID NO:30, 31, 32, respectively. NO:30, 31, and 32 have amino acid sequences in three CDR regions with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity, respectively.

[0008] In some embodiments, the antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:33, or has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:33.

[0009] In the context of this invention, the term "antibody" is used in the broadest sense and explicitly covers single-domain antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, and multispecific antibodies (e.g., bivalent antibodies) formed from at least two intact antibodies, provided they exhibit the desired biological activity.

[0010] In this invention, the modified antibody sequence also falls within the scope of protection of this invention. The term "modification" refers to any form of modification to an amino acid sequence, such as substitution, deletion, insertion, and / or addition of amino acids. The term "substitution" refers to replacing one or more amino acids in the original amino acid sequence with a different amino acid. The term "deletion" refers to reducing one or more amino acids in the original amino acid sequence. The terms "insertion" or "addition" refer to changes in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence.

[0011] In this invention, the modification preferably occurs in regions other than the variable region, such as the constant region or frame region of the antibody, and the modified antibody still retains the desired functional properties of the antibody or its antigen-binding fragment of this invention, or has improved antigen-binding properties.

[0012] The term "identity," also known as "homology," refers to a sequence that is at least 80% identical in amino acid sequence to the sequence provided in this invention. To determine sequence identity, sequence alignment can be performed using various methods known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR), Snapgene, or DNAMAN software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required to achieve optimal alignment across the full-length sequences being compared.

[0013] In this invention, the CDR area is defined according to the IMGT numbering system.

[0014] Unless otherwise stated, the antibodies described herein are isolated antibodies. The term "isolated" as used herein means nucleic acids or antibodies or fragments thereof that have been extracted from their natural environment. "Isolated" nucleic acids or antibodies or fragments thereof therefore include nucleic acids or antibodies or fragments thereof purified by standard purification methods. The term also includes nucleic acids or antibodies or fragments thereof prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids and / or antibodies.

[0015] The second aspect of the present invention provides a humanized antibody that targets and binds to GPRC5D. The humanized antibody is obtained by humanizing residues at key positions in the amino acid sequence frame region shown in SEQ ID NO:16, SEQ ID NO:33 or SEQ ID NO:22 with reference to a human germline frame.

[0016] In some embodiments, the humanized antibody includes any one of B05HM1, B05HM2, B05HM3, B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, and C07HS3.

[0017] In some embodiments, B05HM1, B05HM2, B05HM3, B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, and C07HS3 respectively have amino acid sequences as shown in SEQ ID NO:17-19 and 97-105.

[0018] In some embodiments, the humanized antibody has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17-19, 97-105.

[0019] A third aspect of the present invention provides a tandem antibody targeting GPRC5D, wherein the tandem antibody is formed by sequentially tandemly connecting the humanized antibodies described in the second aspect.

[0020] In some embodiments, the tandem configuration antibody further includes a linker protein.

[0021] In some embodiments, the linker protein is a flexible polypeptide with the structure (G4S). n (n = 2-6), with a total length of 10–30 amino acids.

[0022] In some embodiments, the linker protein is (G4S)3, whose amino acid sequence is shown in SEQ ID NO:114.

[0023] In some embodiments, the tandem antibody configuration is formed by sequentially linking a humanized antibody, a linker protein, and a humanized antibody.

[0024] In some embodiments, the humanized single-domain antibody is selected from any two of B05HS2, F08HS2, and C07HS2.

[0025] In an optional embodiment, the humanized single-domain antibody is formed by sequentially linking B05HS2, a linker protein, and F08HS2.

[0026] In an optional embodiment, the humanized single-domain antibody is formed by sequentially linking C07HS2, a linker protein, and B05HS2.

[0027] In an optional embodiment, the humanized single-domain antibody is formed by sequentially linking F08HS2, a linker protein, and C07HS2.

[0028] In a specific embodiment of the present invention, the tandem configuration antibody has an amino acid sequence as shown in SEQ ID NO:106, 107, 108, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:106, 107, 108.

[0029] A fourth aspect of the present invention provides a chimeric antigen receptor comprising the antibody described in the first aspect, the humanized antibody described in the second aspect, or the tandem antibody described in the third aspect.

[0030] In some embodiments, the chimeric antigen receptor further includes a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signal transduction region.

[0031] In some embodiments, the chimeric antigen receptor further includes an extracellular signal peptide CD8 SP, the amino acid sequence of which is shown in SEQ ID NO:36.

[0032] In some embodiments, the chimeric antigen receptor further includes EGFRt, the amino acid sequence of which is shown in SEQ ID NO:43.

[0033] In some embodiments, the chimeric antigen receptor further includes an EGFRt signal peptide, the amino acid sequence of which is shown in SEQ ID NO:42.

[0034] In some embodiments, the chimeric antigen receptor further includes a T2A linker, the amino acid sequence of which is shown in SEQ ID NO:41.

[0035] In an optional embodiment, the hinge region is selected from the hinge regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof.

[0036] In optional embodiments, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof.

[0037] In an optional embodiment, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domains of the following molecules: 41BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof.

[0038] In an optional embodiment, the intracellular signal transduction region is selected from the intracellular signal transduction regions of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d, ZAP70, and variants thereof.

[0039] In a specific embodiment of the present invention, the hinge region is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:37.

[0040] In a specific embodiment of the present invention, the transmembrane region is the CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:38.

[0041] In a specific embodiment of the present invention, the co-stimulatory signaling domain is a 4 1BB co-stimulatory signaling domain, and the amino acid sequence of the 4 1BB co-stimulatory signaling domain is shown in SEQ ID NO:39.

[0042] In a specific embodiment of the present invention, the intracellular signal transduction region is the CD3ζ intracellular signal transduction region, and the amino acid sequence of the CD3ζ intracellular signal transduction region is shown in SEQ ID NO:40.

[0043] In some embodiments, those skilled in the art may change the combination of signal peptide, extracellular hinge region and transmembrane domain, co-stimulatory domain and intracellular immune receptor tyrosine activation motif according to actual conditions or needs. Regardless of the form of change, as long as the chimeric antigen receptor has the CDR sequence or heavy chain variable region of the antibody or humanized antibody described in this invention, it falls within the protection scope of this invention.

[0044] In a specific embodiment of the present invention, the chimeric antigen receptor is formed by sequentially connecting the extracellular signal peptide CD8 SP, the antibody described in the first aspect or the humanized antibody described in the second aspect or the tandem antibody described in the third aspect, the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signal domain, the CD3ζ intracellular signal transduction region, the T2A linker, the EGFRt signal peptide, and EGFRt.

[0045] The fifth aspect of the present invention provides an immune effector cell that expresses the chimeric antigen receptor described in the fourth aspect.

[0046] In some embodiments, the immune effector cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages.

[0047] In an optional embodiment, the immune effector cells are T cells.

[0048] The sixth aspect of the present invention provides a pharmaceutical composition comprising the antibody described in the first aspect, the humanized antibody described in the second aspect, the tandem antibody described in the third aspect, or the immune effector cells described in the fifth aspect.

[0049] In this invention, the term "pharmaceutical composition" refers to a composition containing at least one bioactive compound. The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. The pharmaceutical compositions of this invention may contain any commonly used non-toxic, pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided the target tissue can be reached.

[0050] The pharmaceutical compositions of the present invention can also be used in combination with other drugs for treating solid tumors. These other compounds for treating solid tumors can be administered simultaneously with the main active ingredient (e.g., the antibody described in the first aspect of the invention), or even simultaneously in the same composition. Other therapeutic compounds can also be administered alone as a single composition or in a dosage form different from that of the main active ingredient.

[0051] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, or buffer solution.

[0052] In this invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise fluids such as water, saline, glycerol, and ethanol. Such carriers may also contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0053] In an optional embodiment, the pharmaceutical composition is an injectable cell preparation.

[0054] The seventh aspect of the present invention provides a biomaterial comprising any one of the following:

[0055] 1) A nucleic acid molecule comprising a nucleotide sequence encoding the antibody described in the first aspect, the humanized antibody described in the second aspect, the tandem antibody described in the third aspect, or the chimeric antigen receptor described in the fourth aspect.

[0056] In this invention, the term "nucleic acid molecule" refers to any polymeric form of any length and composed of ribonucleotides or deoxyribonucleotides. Typically, a nucleic acid molecule is a coding sequence, as used herein, referring to a DNA sequence that, when placed under the control of a suitable regulatory sequence, is transcribed and translated into a polypeptide in a host cell. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences may include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even recombinant DNA sequences. The transcription stop sequence will typically be located at the 3' end of the coding sequence.

[0057] In some embodiments, the nucleotide sequence is as shown in SEQ ID NO:45-60, 109-111 or is a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:45-60, 109-111.

[0058] 2) A recombinant expression vector comprising the nucleic acid molecule described in 1).

[0059] In this invention, the terms "recombinant expression vector," "vector," and "expression vector" are used interchangeably to refer to an artificial construct capable of delivering and preferentially expressing one or more target genes or sequences in a host cell. The vector can be a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, herpesvirus vectors, and other viral vectors. Other viral vectors may include bacteriophage vectors, baculovirus vectors, animal viral vectors, plant viral vectors, and may include papillomavirus vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, EB virus vectors, retroviral vectors, etc.

[0060] In some embodiments, the recombinant expression vector is suitable for in vitro or in vivo delivery systems, including viral vectors, lipid nanoparticles (LNPs), or other delivery vectors capable of delivering and expressing the nucleic acid molecule in a subject.

[0061] 3) Recombinant host cells, wherein the recombinant host cells express the antibodies described in the first aspect, the humanized antibodies described in the second aspect, the tandem antibody described in the third aspect, or the chimeric antigen receptor described in the fourth aspect.

[0062] The recombinant host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. The host cell includes any progeny of the parent cell that differs from the parent cell due to mutations occurring during replication. Preferably, the recombinant host cell includes prokaryotic cells and eukaryotic cells; more preferably, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae; even more preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; most preferably, the recombinant host cell is an immune cell; most preferably, the immune cell includes T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, or any combination thereof; most preferably, the immune cell is a T cell.

[0063] In this invention, the term "T cell" refers to a major type of leukocyte that matures in the thymus and plays various roles in the immune system, including identifying specific foreign antigens in the body and activating or inactivating other immune cells. T cells can be any type of T cell, such as cultured T cells, for example, primary T cells, or T cells derived from cultured T cell lines (e.g., Jurkat, SupT1, etc.) or derived from mammals. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any developmental stage, including but not limited to: CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, primary T cells, regulatory T cells, γδ T cells, etc. Other types of helper T cells include cells such as Th3, Th17, Th9, or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tern cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as proprietary T cell receptor fusion constructs (TRuCs) modified to express T cell receptors (TCRs), chimeric antigen receptors (CARs), synthetic T cell receptors and antigen receptors (STARs), or TCR2.

[0064] In some embodiments, the recombinant host cell comprises the recombinant expression vector described in 2).

[0065] In some embodiments, the recombinant host cell comprises recombinant immune cells.

[0066] In an optional embodiment, the recombinant immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages.

[0067] In a specific embodiment of the present invention, the recombinant immune cells are T cells.

[0068] The eighth aspect of the present invention provides an antibody derivative, which includes the antibody described in the first aspect, the humanized antibody described in the second aspect, or the tandem antibody described in the third aspect and a detectable marker conjugated thereto.

[0069] In some embodiments, the detectable marker includes at least one of a radioactive isotope, a metallic nanomaterial, fluorescein, biotin, avidin, a biotin / avidin protein complex, a biotin / avidin protein complex, a chromophore, an electron-dense substance, and an enzyme.

[0070] The ninth aspect of the present invention provides any of the following applications, the applications including:

[0071] 1) The use of the antibody described in the first aspect, the humanized antibody described in the second aspect, the tandem antibody described in the third aspect, or the antibody derivative described in the eighth aspect in the detection of GPRC5D protein or its antigen fragment.

[0072] 2) The use of the antibody described in the first aspect, the humanized antibody described in the second aspect, the tandem antibody described in the third aspect, or the antibody derivative described in the eighth aspect in the preparation of products for detecting GPRC5D protein or its antigen fragments.

[0073] 3) The use of the antibodies described in the first aspect, the humanized antibodies described in the second aspect, or the tandem antibodies described in the third aspect in constructing chimeric antigen receptors or preparing immune effector cells.

[0074] 4) The application of the nucleic acid molecule or the recombinant expression vector described in the seventh aspect in the preparation of recombinant host cells, wherein the recombinant host cells are the recombinant host cells described in the seventh aspect.

[0075] 5) The use of the antibodies described in the first aspect, the humanized antibodies described in the second aspect, the tandem antibodies described in the third aspect, the chimeric antigen receptors described in the fourth aspect, the immune effector cells described in the fifth aspect, the pharmaceutical compositions described in the sixth aspect, and the biomaterials described in the seventh aspect in the preparation of drugs for treating GPRC5D positive tumors.

[0076] In some embodiments, the medicament is a therapeutic agent based on immune cells or an in vivo delivery system, including recombinant immune cells (such as CAR-T, CAR-NK, TruC-T, STAR-T) prepared ex vivo or in vivo expression systems.

[0077] In some embodiments, the GPRC5D positive tumor is multiple myeloma.

[0078] The tenth aspect of the present invention is a method comprising:

[0079] 1) A method for producing any one of the antibodies described in the first aspect, the humanized antibodies described in the second aspect, the tandem antibody described in the third aspect, and the chimeric antigen receptor described in the fourth aspect, the method comprising: culturing the recombinant host cells described in the seventh aspect.

[0080] In some embodiments, the antibodies of the present invention are obtained through artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention are obtained through direct amino acid synthesis. In some embodiments, the antibodies of the present invention are obtained through genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Prokaryotic cell expression systems include *Escherichia coli* expression systems. Eukaryotic cell expression systems include enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0081] 2) A method for preparing a recombinant host cell according to the eighth aspect, the method comprising: introducing the recombinant expression vector according to the seventh aspect into a host cell.

[0082] 3) A method for detecting GPRC5D protein or its antigen fragment, the method comprising: contacting a sample to be tested with the antibody described in the first aspect, the humanized antibody described in the second aspect, the tandem antibody described in the third aspect, or the antibody derivative described in the eighth aspect, and detecting the formation of the antigen-antibody complex.

[0083] 4) A method for treating a subject with a GPRC5D-positive tumor, the method comprising: administering to the subject the antibody described in the first aspect, the humanized antibody described in the second aspect, the tandem antibody described in the third aspect, the chimeric antigen receptor described in the fourth aspect, the immune effector cells described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect.

[0084] In some implementations, the administration method includes the reinfusion of immune cells prepared in ex vivo or the delivery of expression systems in vivo.

[0085] In some embodiments, the subject includes mammals; in a specific embodiment of the invention, the subject is preferably a human.

[0086] Advantages and benefits of the present invention: The CARs constructed based on the single-domain antibody, humanized antibody or tandem antibody provided by the present invention can be transduced into T cells to create CAR-T cells that specifically target GPRC5D, providing a new treatment option for patients with GPRC5D-positive multiple myeloma. Attached Figure Description

[0087] Figure 1 shows the hybrid database construction sequence.

[0088] Figure 2 is a schematic diagram of the pcDNA3.4-IgG1Fc vector construction structure.

[0089] Figure 3 shows the cell binding verification diagram of GPRC5D antibody.

[0090] Figure 4 shows the affinity verification diagram of the purified antibody from GPRC5D.

[0091] Figure 5 shows the binding ability of the purified GPRC5D candidate antibody to CHO-S-Mouse-GPRC5D.

[0092] Figure 6 is a schematic diagram of the CAR-T vector construction structure.

[0093] Figure 7 shows the CAR-T killing rate of the GPRC5D candidate purified antibody.

[0094] Figure 8 shows the affinity verification of B05 antibody-humanized FACS with CHO-S-GPRC5D cells.

[0095] Figure 9 shows the affinity of B05 antibody-humanized FACS to CHO-S cells.

[0096] Figure 10 shows the affinity assay of B05 antibody-humanized FACS combined with CHO-Mouse GPRC5D cells.

[0097] Figure 11 shows the CAR-T killing rate detection of the GPRC5D humanized antibody.

[0098] Figure 12 is a schematic diagram of the structure of the humanized antibody CAR.

[0099] Figure 13 shows the results of detecting the killing power of CAR-T cells constructed with humanized antibodies.

[0100] Figure 14 is a schematic diagram of the humanized antibody tandem configuration CAR structure.

[0101] Figure 15 shows the results of detecting the killing power of CAR-T cells constructed using humanized antibody tandem configuration. Detailed Implementation

[0102] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified. Simple modifications to the present invention based on its essence are all within the scope of protection claimed by the present invention.

[0103] Example 1: Construction of a phage display library, antibody screening and validation

[0104] 1. Alpaca immune phage display library construction

[0105] Two alpacas were immunized a total of four times. The immunogen was 293F-GPRC5D (human sequence), administered subcutaneously, with GERBU as the adjuvant. After immunization, serum was isolated from the immunized alpacas and validated by ELISA and FACS. Peripheral blood was then collected from the alpacas, PBMCs were isolated, RNA was extracted, and reverse transcribed into cDNA. Using a single-domain antibody cloning primer combination, the VHH sequence was amplified from the cDNA sample and subcloned into the phage display vector pDisplay. The pDisplay was then electrotransformed into SS320 E. coli competent cells to construct a single-domain antibody phage display library (co-constructed from both alpacas). The sequence composition of the co-constructed library is shown in Figure 1. The co-constructed library from the two alpacas yielded 48 clones. After removing two antibody sequences with a one-base deletion and one empty vector sequence, the remaining sequences were all antibody sequences, indicating good diversity.

[0106] 2. Antibody selection and validation

[0107] In the above-mentioned constructed library, the following screening methods were used: GPRC5D-VLP protein / CHO-S-GPRC5D cell line for screening protein / cell line and VLP protein / CHO-S cell line for screening protein / cell line. Rounds one and three were VLP screening, and rounds two and four were cell screening. Candidate clone sequences 1-B11, 2-D06, 1-G08, 1-G09, 2-A12, 2-B05, 2-B06, and 2-B08 were selected and constructed into the pcDNA3.4-IgG1Fc (SEQ ID NO:35) antibody expression vector (as shown in Figure 2). After transfection with 293F, the supernatant was used to verify binding with CHO-S-GPRC5D cells. The effective binding clone antibodies were purified and then tested for FACS EC50. The results showed that the candidate antibodies bound to CHO-S-GPRC5D cells, with C07, F08, B05, A12, and B11 showing the strongest binding.

[0108] 1) GPRC5D antibody cell binding validation: Flow cytometry detection parameters: using 3×10 5 Cell count was determined per well. The primary antibody was GPRC5D transfection supernatant (100 μl / well), which included positive antibody (10 μg / ml, 100 μl / well); the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). The results are shown in Figure 3.

[0109] 2) GPRC5D purified antibody affinity verification: Flow cytometry parameters: CHO-S-GPRC5D cells (3×10⁻⁶) were used. 5 / well). The primary antibody was GPRC5D target candidate antibody and positive antibody (starting from 30 μg / ml, serially diluted 3 times for 11 spots, 100 μl / well); the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). The results are shown in Figure 4 and Table 1.

[0110] Table 1. Antibody Affinity Analysis Table

[0111] 3) Binding assay of GPRC5D candidate purified antibody with CHO-S-Mouse-GPRC5D: 3×10 CHO-S cells overexpressing mouse GPRC5D were used. 5 Cells were analyzed by flow cytometry using GPRC5D candidate antibody + positive antibody (10 μg / ml, 100 μl / well) as primary antibody and PE-Goat anti-Human IgG Fc (Invitrogen, Cat#: 12-4998-82) (1:1000 dilution) as secondary antibody. The results, shown in Figure 5, indicate that the B05 and F08 candidate antibodies bind strongly to CHO-S-Mouse-GPRC5D cells.

[0112] Example 2: Construction of CAR-T cells from candidate antibody sequences and verification of their killing ability

[0113] 1. CAR Lentiviral Preparation: The positive control CAR-T was obtained by constructing a CAR using BCMA instead of GPRC5D (SEQ ID NO:44, reference doi:10.1158 / 2643-3230.BCD-20-0020). The pCDH-EF1α lentiviral expression plasmid was synthesized, and its structure is shown in Figure 6. The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated, filtered, and stored at -80℃ for later use.

[0114] 2. CAR-T Cell Construction: Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and transduced with the virus within 72 hours of activation. After 24 hours of transduction, the medium was changed and cultured until day 8. Cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify EGFR-t molecules on the surface of T cells, ensuring a positive rate greater than 30%. If the positive rate was lower, EGFR-PE primary antibody and PE magnetic beads were used for enrichment and sorting. CAR-T positive cells were mixed with target cells according to the ratio (effect-to-target ratio 1:1 and 5:1) for subsequent killing experiments, and the killing rate was detected. The results are shown in Figure 7.

[0115] 3. Humanized sequence antibody affinity detection: Based on the original antibody sequence information developed in previous projects, a homology model of the antibody is obtained through modeling, and CDRs are analyzed. The framework amino acids within a specific range, these amino acid sites typically influence the conformation of the CDR or its antigen-binding activity. Human germlines were obtained through IMGT analysis. The selected human germline framework was spliced ​​with the antibody's CDRs, and the framework region sequences of the designed humanized antibody were compared with those of the original antibody. Based on the homology modeling results of the parent antibody, amino acids similar to the surface residues of the human antibody were selected for replacement while maintaining antibody activity and reducing heterology, designing humanized antibody sequences, resulting in B05HM1, B05HM2, and B05HM3 (amino acid sequences shown in SEQ ID NO: 17-19, base sequences shown in SEQ ID NO: 49-51, and base sequences of HCDR1-3 shown in SEQ ID NO: 94-96). The humanized antibodies were then genetically synthesized and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vector was verified by sequencing, endotoxin-free plasmids were prepared and humanized antibodies were expressed in 293F cells. The affinity of the antibodies was then tested after incubation with CHO-S and CHO-S-GPRC5D.

[0116] 1) Affinity test results are shown in Figure 8. The B05 antibody-humanized FACS was validated using CHO-S-GPRC5D cells. CHO-S-GPRC5D cells (3 × 10⁻⁶) were used for the assay. 5 / well), the primary antibody is GPRC5D target candidate antibody and positive antibody (starting from 30μg / ml, 3-fold serial dilution for 10 spots, 100μl / well); the secondary antibody is PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution).

[0117] 2) Humanized FACS using B05 antibody combined with CHO-S cells (as negative cells) for detection, using CHO-S cells (3 × 10⁻⁶ cells). 5The primary antibody for each well was a candidate antibody and a positive antibody targeting GPRC5D (starting at 30 μg / ml, serially diluted 3-fold for 10 spots, 100 μl / well); the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). The results are shown in Figure 9.

[0118] 3) Humanized FACS using B05 antibody combined with CHO-Mouse GPRC5D cells (mouse GPRC5D) for detection. CHO-Mouse GPRC5D cells (3×10⁻⁶) were used. 5 The primary antibody for each well was a candidate antibody and a positive antibody targeting GPRC5D (starting at 30 μg / ml, serially diluted 3-fold for 10 spots, 100 μl / well); the secondary antibody was PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (1:1000 dilution). The results are shown in Figure 10.

[0119] 4. The candidate B05HM2 and B05HM3 sequences were synthesized to construct the pCDH-EF1α lentiviral expression plasmid. The lentiviral system plasmid (pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) was transfected into adherent 293T cells in logarithmic growth phase. The cell culture supernatant was harvested 48-72 hours after transfection, concentrated and filtered to obtain CAR lentivirus, which was stored at -80℃ for later use. Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads, and viral transduction was performed within 72 hours of activation. After 24 hours of transduction, the medium was changed and cultured until day 8. The cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify EGFRt molecules on the surface of T cells, ensuring a positive rate of greater than 30%. If the positive rate was lower, EGFR-PE primary antibody and PE magnetic beads were used for enrichment and sorting. Subsequent killing experiments were conducted by mixing CAR-T positive cells with target cells according to the ratio (effect-to-target ratio of 1:1 and 5:1), and the killing rate was detected. The results are shown in Figure 11.

[0120] Example 3: Different Modification Strategies for Humanized Antibodies and Construction of Tandem Antibodies

[0121] 1. Humanization Design and Sequence Acquisition of VHH Antibodies: This invention uses non-human VHH antibodies as templates. The Abysis online analysis tool (http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) is used to divide the amino acid sequence into domains and annotate key sites, identifying the variable regions FR1-FR4 and CDR1-CDR3. Human antibody templates with the highest homology to these regions are selected as the humanization backbone. Based on this, mutation design is performed according to humanization principles: non-human amino acid residues are replaced with human template residues as much as possible while maintaining antigen-binding ability; conserved structural sites in the FR region are preferentially replaced with homologous amino acids to avoid secondary structure perturbation; in the FR2-FR3 region, if the original amino acid residue is hydrophilic, it is not mutated to a hydrophobic residue to maintain solubility. The sequence humanization results of the three VHH antibodies B05 / C07 / F08 are shown in Table 2, where the T20 score is in parentheses; HS2 is a "conservative strategy" for the FR2 segment, that is, no mutation is performed in this segment to maintain the stability of the antibody backbone.

[0122] Table 2. Humanized antibody sequences

[0123] 2. Construction of Humanized Antibody for CAR-T and Detection of Killing Power: Lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmid) were constructed based on the above sequences. The CAR structure is shown in Figure 12. The obtained plasmids were co-transfected into logarithmically growing adherent 293T cells using a four-plasmid lentiviral system (pCDH-EF1α expression vector, PsPAX2, pMD2.G, and helper plasmid pRev, mixed in a mass ratio of 4:3:2:1). Cell supernatants were collected after 48–72 hours, concentrated, filtered, and lentivirus was obtained and stored at -80℃ for later use. Peripheral blood was collected from patients or healthy volunteers via leukoablation, and PBMCs were obtained by Ficoll density gradient centrifugation. EasySep was used to analyze the leukocytes. TM Human T Cell Isolation Kit (STEMCELL, #17951) for T cell isolation. It is formulated with 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult. TMAfter normal activation of T cells with the human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibody in X-vivo (lonza) medium for 2-3 days, lentivirus transduction was used. The medium was then replaced with one lacking the activating antibody, and the cells were amplified to 9-11 days. The CAR-T cell positivity rate was measured, and the positivity rate of each group was adjusted to a consistent level by mixing in T cells from the same batch. Killing ability was verified by co-incubating with K562 target cells overexpressing GPRC5D at effector-to-target ratios of 1:1 and 1:10 for 16 hours. Humanized CAR-T cells showed good killing ability. Killing ability was verified by co-incubating with K562 target cells not expressing GPRC5D at an effector-to-target ratio of 1:1, and no non-specific killing phenomenon was observed (Figure 13).

[0124] 3. Construction of CAR-T Cells with Tandem Antibodies and Detection of Killing Power: Humanized HS2 clones targeting the FR2 segment using a "conservative strategy" were tandemly constructed in pairs to obtain new sequences (amino acid sequences are shown in Table 3, and the base sequences corresponding to SEQ ID NO:106-108 are shown in SEQ ID NO:109-111). Lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmid) were then constructed, and the CAR structure is shown in Figure 14. The obtained plasmids were co-transfected into logarithmically growing adherent 293T cells using a four-plasmid lentiviral system (pCDH-EF1α expression vector, PsPAX2, pMD2.G, and helper plasmid pRev, mixed in a mass ratio of 4:3:2:1). Cell supernatants were collected after 48-72 hours, concentrated, filtered, and lentivirus was obtained and stored at -80℃ for later use. Peripheral blood was collected from patients or healthy volunteers via leukoablation, and PBMCs were obtained by Ficoll density gradient centrifugation. EasySep was used to analyze the leukocytes. TM Human T Cell Isolation Kit (STEMCELL, #17951) for T cell isolation. It is formulated with 10 ng / mL IL-7 (nearshore protein, GMP-C086), 5 ng / mL IL-15 (nearshore protein, GMP-C016), and ImmunoCult. TMAfter normal activation of T cells using the Human CD3 / CD28 / CD2 T Cell Activator (STEMCELL, #10970) antibody in X-vivo (lonza) medium for 2-3 days, lentivirus transduction was performed. The medium was then replaced with one lacking the activating antibody, and the cells were amplified to 9-11 days. The CAR-T cell positivity rate was measured, and the positivity rate of each group was adjusted to a consistent level by mixing in T cells from the same batch. Using 109 clones (referencing patent: US10633426) and F08HS2 clones as positive controls, CAR-T cells were co-incubated with K562 target cells overexpressing GPRC5D at effector-to-target ratios of 1:1 and 1:5 for 16 hours to verify cytotoxicity. The tandem antibody-constructed CAR-T cells showed higher targeted killing ability. Co-incubation at an effector-to-target ratio of 1:1 with K562 target cells not expressing GPRC5D showed no non-specific killing phenomenon (Figure 15).

[0125] Table 3. Tandem configuration antibodies and CAR sequences

[0126] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An antibody that targets and binds to GPRC5D, characterized in that, The antibody has the amino acid sequences of the three CDR regions shown in SEQ ID NO:1, 2, 3; SEQ ID NO:5, 6, 7; SEQ ID NO:9, 10, 11; SEQ ID NO:13, 14, 15; SEQ ID NO:9, 20, 21; SEQ ID NO:23, 24, 25; SEQ ID NO:27, 28, 25; SEQ ID NO:30, 31, 32, or has the same amino acid sequences as those shown in SEQ ID NO:1, 2, 3; SEQ ID NO:5, 6, 7; SEQ ID NO:9, 10, 11; SEQ ID NO:13, 14, 15; SEQ ID NO:9, 20, 21; SEQ ID NO:23, 24, 25; SEQ ID NO:27, 28, 25; SEQ ID NO:30, 31, 32. NO:30, 31, and 32 have amino acid sequences in three CDR regions with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity, respectively. Preferably, the antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29 or SEQ ID NO:33, or has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:

33.

2. A humanized antibody that targets and binds to GPRC5D, characterized in that, The humanized antibody is obtained by humanizing residues at key positions in the amino acid sequence frame region shown in SEQ ID NO:16, SEQ ID NO:33 or SEQ ID NO:22 with reference to a human germline frame. Preferably, the humanized antibody includes any one of B05HM1, B05HM2, B05HM3, B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, and C07HS3. Preferably, B05HM1, B05HM2, B05HM3, B05HS1, B05HS2, B05HS3, F08HS1, F08HS2, F08HS3, C07HS1, C07HS2, and C07HS3 have amino acid sequences as shown in SEQ ID NO:17-19 and 97-105, respectively. Preferably, the humanized antibody has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17-19, 97-105.

3. A tandem conformation antibody targeting GPRC5D, characterized in that, The tandem antibody configuration is formed by sequentially tandemly connecting the humanized antibodies described in claim 2; Preferably, the tandem antibody further includes a linker protein; Preferably, the linker protein is a flexible polypeptide with the structure (G4S). n (n = 2-6), with a total length of 10–30 amino acids; Preferably, the linker protein is (G4S)3, and its amino acid sequence is shown in SEQ ID NO:114; Preferably, the tandem antibody is formed by sequentially linking a humanized antibody, a linker protein, and a humanized antibody. Preferably, the humanized single-domain antibody is selected from any two of B05HS2, F08HS2, and C07HS2; More preferably, the humanized single-domain antibody is formed by sequentially linking B05HS2, a linker protein, and F08HS2; More preferably, the humanized single-domain antibody is formed by sequentially linking CO7HS2, a linker protein, and B05HS2; More preferably, the humanized single-domain antibody is formed by sequentially linking F08HS2, a linker protein, and C07HS2; Most preferably, the tandem antibody has an amino acid sequence as shown in SEQ ID NO:106, 107, 108, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:106, 107, 108.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the antibody of claim 1, the humanized antibody of claim 2, or the tandem antibody of claim 3; Preferably, the chimeric antigen receptor further includes a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signal transduction region; Preferably, the chimeric antigen receptor further includes the extracellular signal peptide CD8 SP, the amino acid sequence of which is shown in SEQ ID NO:36; Preferably, the chimeric antigen receptor further includes EGFRt, the amino acid sequence of which is shown in SEQ ID NO:43; Preferably, the chimeric antigen receptor further includes an EGFRt signal peptide, the amino acid sequence of which is shown in SEQ ID NO:42; Preferably, the chimeric antigen receptor further includes a T2A linker, the amino acid sequence of which is shown in SEQ ID NO:41; More preferably, the hinge region is selected from the hinge regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and variants thereof; More preferably, the transmembrane region is selected from the transmembrane regions of the following molecules: CD8, 41BB, IgG1, IgG4, PD1, CD28, CD34, OX40, CD3ε, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and variants thereof; More preferably, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domains of the following molecules: 41BB, HVEM, CD27, CD19, CD28, ICOS, CD4, CD8α, CD8β, CD40, OX40, DR3, CD2, GITR, CD30, TIM1, CD226, CD278 and variants thereof; More preferably, the intracellular signal transduction region is selected from the intracellular signal transduction regions of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, CD278, CD21, CD22, FcεRI, FcRγ, FcRβ, CD4, CD5, CD8, CD79a, CD79b, DAP10, DAP12, CD66d, ZAP70 and their variants; Most preferably, the hinge region is a CD8 hinge region, and the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO:37; Most preferably, the transmembrane region is a CD8 transmembrane region, and the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO:38; Most preferably, the co-stimulatory signaling domain is a 4 1BB co-stimulatory signaling domain, and the amino acid sequence of the 4 1BB co-stimulatory signaling domain is shown in SEQ ID NO:39; Most preferably, the intracellular signal transduction region is the CD3ζ intracellular signal transduction region, and the amino acid sequence of the CD3ζ intracellular signal transduction region is shown in SEQ ID NO:40; Most preferably, the chimeric antigen receptor is formed by sequentially connecting the extracellular signal peptide CD8 SP, the antibody of claim 1 or the humanized antibody of claim 2 or the tandem antibody of claim 3, the CD8 hinge region, the CD8 transmembrane region, the 41BB co-stimulatory signaling domain, the CD3ζ intracellular signal transduction region, the T2A linker, the EGFRt signal peptide, and EGFRt.

5. Immune effector cells, characterized in that, The immune effector cells express the chimeric antigen receptor as described in claim 4; Preferably, the immune effector cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages; More preferably, the immune effector cells are T cells.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, or the immune effector cell of claim 5; Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, or buffer solution; More preferably, the pharmaceutical composition is a cell preparation for injection.

7. A biomaterial, characterized in that, The biomaterial includes any one of the following: 1) A nucleic acid molecule comprising a nucleotide sequence encoding the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, or the chimeric antigen receptor of claim 4; Preferably, the nucleotide sequence is as shown in SEQ ID NO:45-60, 109-111 or is a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:45-60, 109-111; 2) A recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid molecule described in 1); Preferably, the recombinant expression vector is suitable for in vitro or in vivo delivery systems, including viral vectors, lipid nanoparticles, or other delivery vectors capable of delivering and expressing the nucleic acid molecules in a subject; 3) Recombinant host cells, wherein the recombinant host cells express the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, or the chimeric antigen receptor of claim 4; Preferably, the recombinant host cell comprises the recombinant expression vector described in step 2); Preferably, the recombinant host cell comprises recombinant immune cells; More preferably, the recombinant immune cells are T cells, NK cells, iNKT cells, B cells, CTL cells, monocytes, myeloid cells, dendritic cells, or macrophages; Most preferably, the recombinant immune cells are T cells.

8. An antibody derivative, characterized in that, The antibody derivatives include the antibody of claim 1, the humanized antibody of claim 2, or the tandem antibody of claim 3 and the detectable marker conjugated thereto; Preferably, the detectable marker includes at least one of the following: radioactive isotopes, metal nanomaterials, fluorescein, biotin, avidin, biotin / avidin protein complex, biotin / avidin protein complex, chromophores, electron-dense substances, and enzymes.

9. Any of the following applications, characterized in that, The applications include: 1) The use of the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, or the antibody derivative of claim 8 in the detection of GPRC5D protein or its antigen fragment; 2) The use of the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, or the antibody derivative of claim 8 in the preparation of products for detecting GPRC5D protein or its antigen fragments; 3) The use of the antibody of claim 1, the humanized antibody of claim 2, or the tandem antibody of claim 3 in constructing chimeric antigen receptors or preparing immune effector cells; 4) The use of the nucleic acid molecule as described in claim 7 or the recombinant expression vector as described in claim 7 in the preparation of recombinant host cells, wherein the recombinant host cells are the recombinant host cells as described in claim 7; 5) The use of the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, the chimeric antigen receptor of claim 4, the immune effector cell of claim 5, the pharmaceutical composition of claim 6, and the biomaterial of claim 7 in the preparation of a medicament for treating GPRC5D positive tumors; Preferably, the drug is a therapeutic agent based on immune cells or an in vivo delivery system, including recombinant immune cells prepared by ex vivo or an in vivo expression system; Preferably, the GPRC5D positive tumor is multiple myeloma.

10. The following method, characterized in that, The method includes: 1) A method for producing any one of the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, and the chimeric antigen receptor of claim 4, the method comprising: culturing the recombinant host cells of claim 7; 2) A method for preparing recombinant host cells according to claim 8, the method comprising: introducing the recombinant expression vector according to claim 7 into host cells; 3) A method for detecting GPRC5D protein or its antigen fragment, the method comprising: contacting a sample to be tested with the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, or the antibody derivative of claim 8, and detecting the formation of the antigen-antibody complex; 4) A method for treating a subject with a GPRC5D-positive tumor, the method comprising: administering to the subject the antibody of claim 1, the humanized antibody of claim 2, the tandem antibody of claim 3, the chimeric antigen receptor of claim 4, the immune effector cells of claim 5, or the pharmaceutical composition of claim 6; Preferably, the administration method includes an ex vivo-prepared immune cell reinfusion system or an in vivo delivery expression system.

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